IJC International Journal of Cancer

The human PKP2/-2 is induced by Wnt/b- in normal and colon cancer-associated fibroblasts

Nuria Niell1,2, Marıa Jesus Larriba1,3,4, Gemma Ferrer-Mayorga1,3,4,5, Isabel Sanchez-P erez 1,2,3,6,7, Ramon Cantero3,8, Francisco X. Real4,9,10, Luis del Peso1,2,3,11, Alberto Munoz~ 1,3,4 and Jose Manuel Gonzalez-Sancho 1,2,4

1 Instituto de Investigaciones Biomedicas “Alberto Sols”, Consejo Superior de Investigaciones Cientıficas (CSIC) –Universidad Autonoma de Madrid (UAM), Madrid, E-28029, Spain 2 Departamento de Bioquımica, Facultad de Medicina, Universidad Autonoma de Madrid (UAM), Madrid, E-28029, Spain 3 Instituto de Investigacion Sanitaria Hospital Universitario La Paz (IdiPAZ), Madrid, E-28046, Spain 4 Instituto de Salud Carlos III, CIBER de Cancer (CIBERONC), Madrid, Spain 5 Fundacion de Investigacion HM Hospitales, Madrid, E-28015, Spain 6 Unidad asociada de Biomedicina UCLM-CSIC 7 Instituto de Salud Carlos III, CIBER de Enfermedades Raras (CIBERER), Madrid, Spain 8 Department of Surgery, La Paz University Hospital, Colorectal Unit, Madrid, E-28046, Spain 9 Cancer Cell Biology Programme, Spanish National Cancer Research Centre, Epithelial Carcinogenesis Group, Madrid, E-28029, Spain 10 Departament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, E-08003, Spain 11 Instituto de Salud Carlos III, CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main com- ponent of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/b- catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibro- blasts. Wnt3A regulates the expression of 1,136 , of which 662 are upregulated and 474 are downregulated in CCD- 18Co cells. A set of genes encoding inhibitors of the Wnt/b-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal Plakophilin-2 is a novel direct transcriptional target of Wnt/b-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by b-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/b-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/b-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the Molecular Cancer Biology feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells.

Key words: colon cancer, normal and cancer-associated fibroblasts, Wnt/b-catenin signalling, PKP2/Plakophilin-2, gene regulation Abbreviations: CAF: cancer-associated fibroblasts; CMS: consensus molecular subtype; CRC: colorectal cancer; ECM: extracellular matrix; EMT: epithelial-mesenchymal transition; NF: normal fibroblasts; PCF: pericryptal fibroblasts; S.D.: standard deviation; TSS: tran- scription start site Additional Supporting Information may be found in the online version of this article. Grant sponsor: Consejerıa de Educacion, Juventud y Deporte, Comunidad de Madrid; Grant number: S2010/BMD-2344 Colomics2; Grant sponsor: Spanish Ministerio de Economıa y Competitividad-Fondos FEDER; Grant numbers: Nurcamein SAF-2015-71878-REDT, SAF2013- 43468-R, SAF2014-53819-R; Grant sponsor: Instituto de la Salud Carlos III - Fondos FEDER; Grant number: CB16/12/00273 CIBERONC, RD12/0036/0021; Grant sponsor: Agencia Estatal de Investigacion - Fondos FEDER; Grant number: SAF2016-76377-R DOI: 10.1002/ijc.31104 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. History: Received 2 Mar 2017; Accepted 4 Oct 2017; Online 16 Oct 2017 Correspondence to: Jose M. Gonzalez-Sancho, Instituto de Investigaciones Biomedicas “Alberto Sols”, C/Arturo Duperier 4, 28029 Madrid, Spain, Tel: (134) 915854457, Fax: (134) 915854401, E-mail: [email protected]

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Niell et al. 793

What’s new? Cancer-associated fibroblasts in the tumour microenvironment play a critical role in colorectal cancer (CRC) progression. In this transcriptomic study of human colon fibroblasts, profound changes in fibroblast gene expression profile were linked to Wnt3A signalling. Wnt3A signalling was associated particularly with the induction of inhibitors of the Wnt/b-catenin pathway, indicative of an inhibitory feedback mechanism. Among the genes most strongly upregulated was PKP2, a novel transcriptional target of Wnt/b-catenin signalling in both normal and colon cancer-associated fibroblasts. Plakophilin-2 protein, previously known to be widely expressed in carcinomas, was further found to antagonize Wnt/b-catenin transcriptional activity.

Colorectal cancer (CRC) is one of the leading causes of this b-catenin destruction complex. In the absence of Wnt cancer-related death in Western countries.1,2 CRC results from ligands, CK1 and GSK3b catalyse b-catenin N-terminal the malignant transformation of epithelial cells lining the lumi- phosphorylation which triggers b-catenin ubiquitination and nal surface of the colonic crypt, a process driven by cumulative subsequent degradation by the proteasome. Inhibition of the b- mutations in both oncogenes and tumour suppressor genes.3,4 catenin destruction complex in response to Wnt signalling Aberrant activation of the Wnt/b-catenin pathway is a hall- results in b-catenin accumulation in the cytoplasm and its mark of colorectal carcinoma and plays a key role in initiation translocation into the nucleus, where it behaves as a transcrip- and progression of the tumour.5 Nevertheless, it is now well- tional co-activator for LEF/TCF transcription factors. The LEF/ accepted that the surrounding tumour stroma plays an essen- TCF family is composed of four members (TCF-1 to 24) that tial role in CRC progression.6–9 Therefore, understanding the bind to b-catenin/TCF binding sites in promoters and reciprocal interaction between colon carcinoma cells and enhancers of target genes and regulate their expression.5 Aber- cancer-associated fibroblasts (CAF) and other cell types in the rant activation of the Wnt/b-catenin pathway is thought to be tumour stroma is of key importance in the design of new, the initial event and a driving force of colorectal tumorigenesis, more efficient strategies against this neoplasia. and most human CRC carry mutations in genes that encode Tumour stroma is composed of extracellular matrix intracellular members of this pathway (including APC, (ECM) and a variety of cell types including endothe- CTNNB1/b-catenin and AXIN1/2 genes).5 lial cells, pericytes, infiltrating immune cells, adipocytes and In spite of the abundant literature on Wnt/b-catenin sig- CAF. A recent collaborative study has classified CRC into nalling in CRC and colon epithelial cells, studies are lacking four consensus molecular subtypes (CMS). CMS4, the type on how Wnt signalling affects colon PCF. It is highly likely with the worst prognosis, is characterized by strong trans- that Wnt factors secreted by PCF and possibly other crypt forming growth factor (TGF)-b activation and high stromal cell types bind not only Wnt receptors in crypt epithelial component.8 Another study has revealed that the CMS4 sub- cells, but also those in PCF themselves, triggering a Wnt sig- type displays high expression of immune signatures with nalling cascade. Therefore, we studied the transcriptomic

overexpression of markers of lymphocytes and cells of mono- response to Wnt3A in established human normal colonic Molecular Cancer Biology cytic origin with strong angiogenic and inflammatory compo- myofibroblasts (CCD-18Co). To our knowledge, this is the nents.9 Moreover, most genes upregulated by TGF-b in first study that explores the transcriptomic effect of Wnt pro- stem-like/mesenchymal, poor prognosis CRC are expressed teins on human colon myofibroblasts. Our analysis rendered by stromal cells: CAF, leukocytes or endothelial cells.10,11 a total of 1,136 regulated genes, of which 662 were upregu- These data highlight the relevance of tumour stroma for lated and 474 were downregulated. The gene encoding the CRC progression. desmosomal protein Plakophilin-2 (PKP2) was upregulated Pericryptal fibroblasts (PCF) in healthy colon stroma by Wnt3A in CCD-18Co as well as in primary cultures of secrete a variety of molecules, including Wnt factors, that normal fibroblasts (NF) and CAF derived from CRC patients. modulate adjacent epithelial cell growth and differentiation.7 Like b-catenin, are members of the armadillo Secreted Wnt ligands bind their receptors located at the family that are located both in the cytoplasm and in the plasma membrane of stem cells and proliferative progenitors nucleus.12 Here we show that PKP2 is a b-catenin/TCF target at the bottom of the colon crypt, and trigger a signalling cas- gene whose expression is regulated through several b-cate- cade that regulates gene expression in the nucleus. The main nin/TCF binding sites present in its promoter and in an effector of this pathway is b-catenin. Wnt binding to Frizzled enhancer sequence located 20 kb upstream from its transcrip- and low density lipoprotein (LDL) receptor-related protein tion start site (TSS). Moreover, our data suggest that (LRP) 5/6 receptor heterodimers leads to phosphorylation of Plakophilin-2 may act as an antagonist of b-catenin/TCF the cytoplasmic tail of LRP and to subsequent inhibition of a complexes on Wnt-activated promoters. multiprotein complex that is responsible for b-catenin degra- dation.5 The tumour suppressors and scaffold proteins APC Material and Methods (encoded by the adenomatous polyposis coli gene) and Axin, Cells and cell culture as well as the kinases Casein Kinase 1 (CK1) and Glycogen CCD-18Co (ATCC CRL-1459) human colon myofibroblasts Synthase Kinase 3b (GSK3b) are the main components of were purchased from the ATCC and cultured in Minimum

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC 794 PKP2 is a novel Wnt/b-catenin target gene in fibroblasts

Essential Medium (MEM, Life Technologies, Carlsbad, CA). using standard parameters. Feature alignments were quanti- IMR-90 fibroblasts (ATCC CCL-186), human embryonic kid- fied using HTSeq-counts from HTSeq framework version ney (HEK)-293T cells, HeLa cells, and MCF7 breast cancer 0.6.0,17 using the GRCh37.75 GTF gene model. We used the cells were cultured in Dulbecco’s modified Eagle’s medium edgeR package version 3.12.118 for R statistical computing (DMEM, Life Technologies). All media were supplemented software to normalize the count data, and statistical analysis with 10% Foetal Bovine Serum (FBS, Life Technologies) and to identify differentially expressed genes. MEM also with L-glutamine and sodium pyruvate (both from Life Technologies). Cell lines were periodically authenticated RNA isolation and RT-qPCR with the GenePrint 10 System (Promega, Fitchburg, WI) and Total RNA was extracted from cells with the RNeasy mini kit the results were sent for comparison against the ATCC cell (Qiagen, Hilden, Germany). Complementary DNA was syn- line database (Manassas, VA). Cells were treated with 200 thesized from 0.5 lg of total RNA using the ImProm-II mg/ml recombinant human Wnt3A (rhWnt3A, R&D Systems, Reverse Transcription System (Promega). Then, the qPCR Minneapolis, MN) in 0.1% phosphate buffer saline–bovine reaction was performed in a CFX384 Real-Time PCR Detec- serum albumin (PBS-BSA) to a final concentration of 100 tion System (Bio-Rad, Hercules, CA), using TaqMan Univer- ng/ml, or with the corresponding vehicle (PBS-BSA) sal Master Mix II without UNG (Applied Biosystems, Foster concentration. City, CA). Thermal cycling of the qPCR reaction was initi- Primary cultures of human colon NF and CAF were ated with a denaturation step at 958C for 10 min, and con- obtained following the explant outgrowth technique13,14 from sisted of 40 cycles (denaturation at 958C 15 sec, annealing fresh surgical specimens of colon primary tumour and mor- and elongation at 608C for 30 sec). We used TaqMan probes phologically normal colonic mucosa (at least 5 cm from the for PKP2 (Hs00428040_m1), PTGER3 (Hs00168755_m1), surgical margin) of the same patient resected from CRC APCDD1 (Hs00537787_m1), DKK2 (Hs00205294_m1), patients subjected to surgery at the Hospital Universitario La NKD1 (Hs00263894_m1), NKD2 (Hs01108239_m1), WNT16 Paz (Madrid, Spain). Samples were provided by the IdiPAZ (Hs00365138_m1), TRPA1 (Hs00175798_m1), ODZ3 (Hs0 Biobank (PT13/0010/0003: Plataforma de Apoyo a la Inves- 1111780_m1), ILDR2 (Hs01025498_m1), AMIGO2 (Hs00 tigacion en Ciencias y Tecnologıas de la Salud en la Red de 827141_g1), PKP1 (Hs00240873_m1), PKP3 (Hs00170 Biobancos 2013), integrated into the Spanish Biobank Net- 887_m1), PKP4 (Hs00269305_m1), VIM (Hs00958111_m1) work (www.redbiobancos.es). Briefly, tissue samples were and ACTA2 (Hs00426835_g1) (Applied Biosystems). The incubated for 30 min with PBS containing 0.5 mg/ml Primo- expression levels were normalized to the levels of human cin (Invivogen, San Diego, CA), 0.1 mg/ml gentamicin and ribosomal protein RPLPO (Taqman probe set 4310879E). All 0.5 mg/ml amphotericin-B (both from Life Technologies). experiments were performed in triplicate. Then, tissue samples were cut into small pieces of approxi- mately 3 mm3 in size and seeded in cell culture flasks in FBS Western blotting

Molecular Cancer Biology with 0.25 mg/ml Primocin. After 1 week and to facilitate Whole-cell extracts were prepared by cell lysis with RIPA fibroblast growth, FBS was replaced by Fibroblast Growth buffer plus protease- and phosphatase-inhibitors for 20 min Medium-2 (FGM-2, Lonza, Basel, Switzerland). Fibroblasts on ice, followed by centrifugation at 13,200 rpm for 15 min grew around the explants for approximately 3 weeks. Then, at 48C. Proteins were separated by SDS-PAGE, transferred to tissue fragments were removed and fibroblasts were routinely PVDF membranes and incubated with antibodies against subcultured at 1:2 ratio in FGM-2. Experiments were per- Plakophilin-2 (sc-136039, Santa Cruz Biotechnology, Santa formed with primary fibroblasts at the seventh passage at Cruz, CA; 651101, Progen, Heidelberg, Germany), b-catenin most. This study was approved by the Ethics Committee for (610154, BD Transduction Laboratories, San Jose, CA), Clinical Research of the Hospital Universitario La Paz GSK3b (610201, BD Transduction laboratories), (HULP-PI-1425), and all patients gave written informed (M7020, clone V9, Dako), a-SMA (A5228, clone 1A4, Sigma- consent. Aldrich), GAPDH (G9545, Sigma-Aldrich) and b- (sc- 1616, Santa Cruz Biotechnology), and then with HRP- RNA sequencing and analysis of gene expression conjugated secondary antibodies. Antibody binding was visu- CCD-18Co cells were stimulated with rhWnt3A or vehicle, alized using the ECL detection system (GE Healthcare, Chal- and three biological replicates per sample were collected and font St. Gills, UK). Films were scanned with a HP Scanjet sequenced on the Illumina HiSeq 2000 platform (Spanish G2710 (Palo Alto, CA) and images were processed using National Cancer Research Centre, CNIO). Image analysis, Adobe Photoshop CS6 (San Jose, CA). Quantification was per-cycle basecalling and quality score assignment was per- done by densitometry, using ImageJ (National Institutes of formed with Illumina Real-Time Analysis software. The Illu- Health, Bethesda, MD). mina2bam tool (Wellcome Trust Sanger Institute—NPG) was used to convert BCL files to bam format. To align fastq PKP2 promoter and enhancer constructs reads, we employed TopHat15 version 2.0.14 against hg19/ Genomic DNA, obtained from CCD-18Co human normal GRCh37.75 assembly of the human reference genome,16 colon myofibroblasts, was used to amplify by PCR two

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Niell et al. 795 upstream sequences of the PKP2 promoter. PCR was per- plasmids harbouring wild-type and mutant PKP2 promoter formed using the Certamp Kit for complex amplifications and enhancer sequences are described above. The expression (Biotools, Madrid, Spain) and primers 50-CATCTCAGCATC plasmid pFLAG-CMV.5.1-Plakophilin2a-flag encoding ATGGTTGG-30 and 50-GAGAAACTCCAACAAGAGGC-30 Plakophilin-2 was purchased from Addgene (Cambridge, for the 21509/–735 fragment, containing the putative b-cate- MA). The expression plasmids pMT23-b-catenin, pCDNA3- nin/TCF binding sites, and 50-TTAATCAACGTTAGC TCF4-VP16 and pCDNA3-p65 have been described else- AGGGC-30 and 50-ACAGGATGGATTTCCGCTCG-30 for where.22–24 The expression plasmid pCDNA3-ICND was a the control 2817/–299 fragment. The resulting PCR products generous gift from Keith R. Brennan (University of Manches- were cloned into the pGEM-T Easy vector (Promega) and ter, UK). A Renilla luciferase plasmid (pRL-TK) was used in sequenced. Subsequently, a second PCR was performed to all experiments as an internal control. Firefly and Renilla generate suitable restriction sites using primers: luciferase activities were measured separately using the Dual 50-TTGGTACCTTCTATTGGGGTGGGAGC-30 and 50- Luciferase reagent kit (Promega) and a GloMax 96 micro- ATAAAGCTTGAGAAACTCCAACAAGAGGC-30 for the plate luminometer (Promega). 21509/–735 fragment, and 50- ATGGTACCTTAATCAA CGTTAGCAGGGC-30 and 50-TACAAGCTTACAGGATGGA Statistical analysis TTTCCGCTCG-30 for the 2817/–299 fragment. The PCR The significance of differences between two groups was products were digested with KpnI and HindIII (New England assessed by the Student’s t test. The differences between more Biolabs, Ipswich, MA), cloned into the pGL4.23 luciferase than two group means were analysed with ANOVA, and reporter plasmid (Promega) and sequenced. Site-directed considered statistically significant when the reported p values mutagenesis of b-catenin/TCF binding sites on the were below 0.05. A Tukey post-hoc test was applied after a 21509/–735 PKP2 promoter construct was performed using significant ANOVA to compare differences between pairs of the Agilent QuikChange II XL Site-Directed Mutagenesis Kit groups. (Agilent Technologies, Santa Clara, CA), and the wild-type 50-CCTTTG(A/T)(A/T)-30 sites were changed into Results 50-CCTTTGGC-30. Primers used for the seven single-site Analysis of Wnt3A transcriptome in CCD-18Co human mutations are shown in Supporting Information Table I. The colon myofibroblasts triple mutant 21509/–735-m347 construct was generated by To investigate the effect of Wnt signalling on human colon three rounds of mutation on the wild-type construct. All con- myofibroblasts, we studied the transcriptional response of structs were sequenced. this cell type to Wnt3A stimulation. For this purpose, we The 220666/–20091 PKP2 enhancer region was amplified selected the colon subepithelial myofibroblast cell line CCD- by PCR using the same template and kit as above, and pri- 18Co25 as a model system. CCD-18Co cells express Frizzled mers 50-CGGTTTTACTCAAGTAAGC-30 and 50- receptors 1 through 7 (Supporting Information Fig. S1A; data 0

GCAATCTTCTTGATGAGTAG-3 . The resulting PCR prod- not shown) and respond to Wnt3A treatment with a substan- Molecular Cancer Biology uct was cloned into the pGEM-T Easy vector and sequenced. tial increase in cytoplasmic levels of b-catenin (Supporting A second PCR was performed to generate KpnI restriction Information Fig. S1B and C). We performed RNA sequenc- sites using primers: 50-TTGGTACCCGGTTTTACTCAA ing on three independent biological replicates of CCD-18Co GTAAGC-30 and 50-ATGGTACCGCAATCTTCTTGATGA cells treated with either Wnt3A (100 ng/ml) or vehicle for 24 GTAG-30. The PCR product was digested with KpnI and hr, as described in the Material and Methods section. An cloned into the pGL4.23 luciferase reporter plasmid. Con- analysis of the data rendered a total of 1,136 differentially structs harbouring the PKP2 enhancer in both forward expressed genes (FDR < 0.05), of which 662 were upregulated (–20666/–20091) and reverse (–20091/–20666) orientations and 474 were downregulated in response to Wnt3A (Sup- were selected and sequenced. Site-directed mutagenesis of porting Information Tables II and III). These genes were b-catenin/TCF binding sites on the 220091/–20666 PKP2 involved in a wide variety of cellular functions. The complete enhancer construct was performed as above and the primers list of genes was deposited in the GEO database (accession used for each single-site mutation are shown in Supporting no. GSE89124). To validate our analysis, we selected some of Information Table I. the upregulated (PTGER3, ODZ3, ILDR2 and AMIGO2) and downregulated (WNT16, TRPA1) genes in our study and per- Transfections, plasmids and reporter assays formed RT-qPCR on a new set of RNA samples. All these Subconfluent cells cultures were transfected in triplicate using genes were confirmed as being regulated by Wnt3A (Fig. 1a). the jetPEI transfection reagent (PolyPlus Transfection, Ill- In support of our study, a gene set enrichment analysis kirch, France), following the manufacturer’s guidelines. The (GSEA) showed that the hallmark gene set that most closely reporter plasmids pTOPFLASH, pFOPFLASH, 4xwtCBF1Luc, correlated with our list of genes regulated by Wnt3A in 4xmtCBF1Luc, NF3, and that harbouring the 22235/1112 CCD-18Co cells was that of genes upregulated by activation region of the DKK1 promoter upstream of the Firefly lucifer- of Wnt signalling through accumulation of CTNNB1/b-cate- ase gene have been previously described.19–22 The reporter nin (FDR q-val 5 0.001; FWER p-val 5 0.001). Interestingly,

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC 796 PKP2 is a novel Wnt/b-catenin target gene in fibroblasts Molecular Cancer Biology

Figure 1. Wnt3A regulates the expression of multiple genes in CCD-18Co human colon myofibroblasts. (a) Validation using RT-qPCR of a representative sample of six genes (PTGER3, ODZ3, ILDR2, AMIGO2, WNT16 and TRPA1) that were up- or downregulated by Wnt3A in RNA- seq experiments. Cells were treated with either Wnt3A (1) or vehicle (–) for 24 hr. The results shown are means 6 S.D. of three indepen- dent biological samples, each one determined in triplicate (***p 0.001). (b) Wnt3A triggers the expression of a set of target genes encod- ing inhibitors of the Wnt/b-catenin pathway. Cells were stimulated with either Wnt3A or vehicle for the indicated times and NKD1, NKD2, DKK2, and APCDD1 expression was analysed by RT-qPCR. Results are shown as fold over control at the indicated times and are means 6 S.D. of three independent biological samples, each one determined in triplicate (n.s., non-significant; *** p 0.001).

the GSEA analysis showed a significant inverse correlation Consequently, genes upregulated in EMT appear to be down- between our list of genes and genes defining epithelial- regulated by Wnt3A in colon myofibroblasts. In contrast, mesenchymal transition (EMT), as in wound healing, fibrosis activation of the Wnt/b-catenin pathway is known to pro- and metastasis (FDR q-val 5 0.022; FWER p-val 5 0.019). mote EMT in epithelial cancer cells.26 Therefore, some

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Niell et al. 797 genetic programs induced by Wnt3A in myofibroblasts differ Both NF and CAF are vimentin and a-SMA positive, and the from those induced in other cell types. This confirms previ- expression of a-SMA is frequently higher in CAF than in ous knowledge that Wnt target genes are cell-type specific. In their paired NF, indicating a more myofibroblastic phenotype agreement, a list of genes upregulated by the EWS-FLI1 of CAF (Supporting Information Fig. S4A and B). Wnt3A oncogenic fusion protein in Ewing’s sarcoma cells showed a consistently increased the expression of PKP2 in both CAF direct correlation with genes upregulated by Wnt3A in CCD- and NF, although with differences among patients (Fig. 3a 18Co cells (FDR q-val < 0.001; FWER p-val < 0.001), and b). although we and others have demonstrated mutual antago- nism between EWS-FLI1 and Wnt/b-catenin signalling in Wnt3A induces PKP2 gene transcription through b-catenin/ Ewing’s sarcoma23,27 (Supporting Information Fig. S2). TCF binding sites To get further insight into the mechanism of PKP2 gene reg- Wnt3A induces the expression of Wnt inhibitors in human ulation by Wnt3A, we treated CCD-18Co cells with actino- fibroblasts mycin D (ACTD), an inhibitor of transcription, before Among the genes upregulated by Wnt3A in CCD-18Co cells, stimulation with Wnt3A. ACTD completely blocked the we detected a set that encodes Wnt/b-catenin pathway inhib- upregulation of PKP2 triggered by Wnt3A (Fig. 4a), which itors, such as the extracellular antagonist Dickkopf-2 (DKK2), argues in favour of a transcriptional mechanism. the membrane-bound inhibitor Adenomatous Polyposis Coli We next made use of the UCSC Genome Browser28 and Downregulated 1 protein (APCDD1), and the cytoplasmic data from the ENCODE Project29 to study the regulatory inhibitors Naked-1 (NKD1), Naked-2 (NKD2), and Axin 2 regions of the human PKP2 gene. Available ChIP-seq data (AXIN2). Time-course experiments confirmed that DKK2 was for transcription factor TCF-4 (TCF7L2 gene) in different cell already upregulated 4 hr after treatment with Wnt3A, while types suggested two putative binding regions for this factor: a NKD1, NKD2 and APCDD1 exhibited significant increases at promoter region around the transcription start site (TSS) of 8 hr post-treatment (Fig. 1b). All these genes were also upre- the human PKP2 gene, and an enhancer region located about gulated by Wnt3A in IMR-90 human lung fibroblasts, which 20 kb upstream from the TSS (Supporting Information Fig. suggests that they may be common Wnt3A targets in fibro- S5A). In silico analysis of both regions revealed up to 7 puta- blasts (Supporting Information Fig. S3). Altogether, these tive b-catenin/TCF binding sites in the promoter (Supporting data suggest that Wnt3A induces the expression of a variety Information Fig. S5B) and up to 3 putative b-catenin/TCF of Wnt inhibitors that, in turn, may be responsible for feed- binding sites in the enhancer (Supporting Information Fig. back inhibition of the pathway in fibroblasts. S5C). We PCR-amplified and cloned both relevant sequences Wnt3A induces PKP2 expression in normal and colon into the pGL4.23 plasmid upstream from the luciferase cancer-associated fibroblasts reporter gene and a minimal promoter (Figs. 4b and 5a). The

For a detailed study, we chose the PKP2 gene, which is con- transcriptional activity of the 21509/–735 PKP2 promoter Molecular Cancer Biology sistently upregulated by Wnt3A in CCD-18Co cells and enco- construct containing all 7 putative b-catenin/TCF binding des the protein Plakophilin-2, whose effect on Wnt signalling sites was consistently stimulated by TCF4-VP16, a constitu- and relation to cancer is controversial (see the Discussion tively active version of TCF-4, in HEK-293T, HeLa and section). Plakophilin-2 belongs to the armadillo-repeat pro- MCF7 cells (Fig. 4c and Supporting Information Fig. S6). Its tein family, along with b-catenin, , p120-catenin, induction was comparable to that of the DKK1 gene pro- and other plakophilins. The PKP2 mRNA level augmented 4- moter, a well-known b-catenin/TCF target gene22,30,31 (Fig. fold in response to Wnt3A (Fig. 2a), which led to a 2-fold 4c). In contrast, a construct containing the 2817/–299 pro- increase in protein level 24 hr after treatment (Fig. 2b). This moter region that does not contain any putative b-catenin/ was due to activation of the Wnt/b-catenin signalling path- TCF binding sites was not activated by TCF4-VP16 (Fig. 4c way, since pre-treatment with the pathway inhibitor XAV- and Supporting Information Fig. S6). Single-site point muta- 939 impaired Plakophilin-2 accumulation (Fig. 2c). Moreover, tions showed that only three putative b-catenin/TCF binding Wnt5A, a noncanonical Wnt that cannot trigger b-catenin- sites contributed significantly to TCF4-VP16 transcriptional induced signalling, could not upregulate PKP2 expression activity (sites #3, #4 and #7; Fig. 4b and d), whereas mutation (Fig. 2d). Other PKP subfamily members, including PKP3 of the remaining motifs (sites #1, #2, #5 and #6) had no and PKP4/p0071, were not regulated by Wnt3A in CCD- effect (data not shown). Finally, a triple mutant targeting 18Co cells, which suggests that the effect on PKP2 is specific these three sites revealed an additive inhibitory effect on (Fig. 2e). TCF4-VP16-mediated transcription, and some lingering To address whether PKP2 upregulation by Wnt3A also activity that suggests the possibility of additional regulatory takes place in primary colonic fibroblasts we established pri- elements or residual activity of mutated sites (Fig. 4b and d). mary cultures of NF and CAF from fresh tumour tissue, and During transcription, enhancer sequences are brought into paired adjacent normal colon mucosa obtained from biopsies close proximity to the TSS, so that transcription factors bind- of CRC patients using the explant outgrowth technique.13,14 ing to these regions can interact with the basal transcriptional

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC 798 PKP2 is a novel Wnt/b-catenin target gene in fibroblasts Molecular Cancer Biology

Figure 2. Wnt3A induces the expression of Plakophilin-2 in CCD-18Co human colon myofibroblasts. (a) RT-qPCR analysis showing the upre- gulation of PKP2 mRNA by Wnt3A. Results are shown as fold over control at the indicated times and are means 6 S.D. of three independent biological samples, each one determined in triplicate (n.s., non-significant; *** p 0.001). (b) Western blot analysis of Plakophilin-2 upre- gulation by Wnt3A in a time course experiment. b-Actin was used as a loading control. Fold increases over vehicle-treated controls are indi- cated. A representative experiment is shown. (c) The Wnt/b-catenin pathway/tankyrase inhibitor XAV-939 reduces Wnt3A-dependent Plakophilin-2 upregulation. Cells were pre-treated for 4 hr with either 1 mM XAV-939 (1) or vehicle (–) and then stimulated with Wnt3A (1) for 24 hr. b-Actin was used as a loading control. A representative experiment is shown. Bar plot represents means 6 S.D. of four indepen- dent experiments (n.s., non-significant; ** p 0.01). (d) Non-canonical Wnt5A cannot induce Plakophilin-2 accumulation. Myofibroblasts were stimulated with either Wnt3A, Wnt5A or both for 24 hr, and Plakophilin-2 expression was analysed by Western blot. b-Actin was used as a loading control. A representative experiment is shown. Bar plot represents means 6 S.D. of four independent experiments (n.s., non- significant; ** p 0.01). (e) RT-qPCR analysis of the expression of PKP family members in response to Wnt3A stimulation. CCD-18Co human colon myofibroblasts were treated with either Wnt3A (1) or vehicle (–) for 24 hr. Results are shown as fold over control and are means 6 S.D. of three independent biological samples, each one determined in triplicate (n.s., non-significant; *** p 0.001).

machinery. We cloned the enhancer sequence described single-point mutations in each of the three putative b-cate- above into pGL4.23 in both forward and reverse orientations nin/TCF binding sites of the reverse-orientated enhancer, and (Fig. 5a), and showed that TCF4-VP16 could stimulate lucif- showed that mutations in site A completely abolished TCF4- erase activity from both constructs with a potency compara- VP16-mediated transcription, while mutations in sites B and ble to that of the DKK1 promoter, and even slightly stronger C were totally innocuous (Fig. 5c). In summary, our data in the reverse orientation (Fig. 5b). Therefore, we generated suggest that b-catenin/TCF complexes induce transcription

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Niell et al. 799 Molecular Cancer Biology Figure 3. Wnt3A induces PKP2/Plakophilin-2 expression in normal fibroblasts (NF) and in cancer-associated fibroblasts (CAF). NF and CAF were obtained from CRC patients as described in the Materials and Methods and treated with either Wnt3A (1) or vehicle (–) for 24 hr. Total RNA and protein was purified and PKP2/Plakophilin-2 mRNA and protein levels were analysed by RT-qPCR (a) and western blot (b), respectively. Results in (a) are shown as fold over the NF control and are means 6 S.D. of three independent biological samples, each one determined in triplicate (**p 0.01; *** p 0.001). b-Actin was used as a loading control in (b) and fold increases over vehicle-treated controls are indicated. of the PKP2 gene through at least 3 binding sites in the pro- expression of Plakophilin-2 in HEK-293T cells impaired moter region and one binding site in an enhancer region activation of the pTOPFLASH reporter by wild-type b-cate- located 20 kb upstream from the TSS (Fig. 5d). nin (Fig. 6a). This effect was specific, since transcriptional activation of the Notch pathway reporter 4xwtCBF1Luc by Plakophilin-2 interferes with b-catenin/TCF transcriptional ICND was unaffected by Plakophilin-2 (Fig. 6b), and activa- activity tion of the NF-jB reporter NF3 by p65 was potentiated, Next, we aimed to address the functional effects that an rather than inhibited, by Plakophilin-2 (Fig. 6c), as previ- increase in PKP2 gene expression triggered by Wnt3A in NF ously described.32 Remarkably, the inhibitory effect of and CAF might have on b-catenin/TCF transcriptional activ- Plakophilin-2 on b-catenin/TCF-mediated transcription was ity. To this end, we used the synthetic pTOPFLASH and also confirmed using the DKK1 promoter construct (Fig. pFOPFLASH plasmids, which contain three copies of either a 6d). This effect was not due to a decrease in b-catenin pro- wild-type or a mutant b-catenin/TCF binding consensus tein level, since this remained unaffected by Plakophin-2 sequence upstream of a minimal c-FOS promoter and the expression (Fig. 6e). Altogether, these data show an inhibi- Firefly luciferase gene, which have been extensively used to tory effect of Plakophilin-2 on b-catenin-responsive study b-catenin/TCF transcriptional activity.19 Transient promoters.

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC 800 PKP2 is a novel Wnt/b-catenin target gene in fibroblasts

Figure 4. The human PKP2 gene promoter is activated by b-catenin/TCF. (a) Actinomycin D (ACTD) blocks Wnt3A-induced accumulation of PKP2 mRNA. CCD-18Co cells were pre-treated for 1 hr with either 5 mg/ml ACTD or vehicle and then stimulated with Wnt3A for 8 hr. Total RNA was purified and PKP2 mRNA levels were analysed by RT-qPCR. Results are shown as fold over control and are means 6 S.D. of three independent biological samples, each one determined in triplicate (n.s., nonsignificant; *** p 0.001). (b) Diagram of the PKP2 promoter constructs used in this study showing the location of the putative wild-type (50-CTTTG[A/T][A/T]-30, white boxes) or mutant (50-CTTTGGC-30, X-crossed white boxes) b-catenin/TCF binding sites. (c) HEK-293T cells were co-transfected with the indicated PKP2 promoter constructs together with TCF4-VP16 (1; a constitutively active form of TCF-4) or an empty vector (–). The b-catenin/TCF responsive DKK1 promoter was used as a positive control. A Renilla luciferase plasmid (pRL-TK) was used as an internal control. Cells were lysed 48 hr after transfection and Firefly and Renilla luciferase activities were measured. Values were normalized to those of Renilla. Error bars represent S.D. (***p 0.001). (d) HEK-293T cells were co-transfected with the indicated PKP2 promoter wild-type or mutant constructs together with TCF4- Molecular Cancer Biology VP16 (1) or an empty vector (–) and the experiment was performed as in (c). Error bars represent S.D. (*p 0.05; ** p 0.01; *** p 0.001).

Discussion are required for stem and proliferating cells at the bottom of Pericryptal stromal cells are essential for the maintenance of the crypt, but could be deleterious for the surrounding fibro- colon tissue homeostasis, and also play an important role in blasts. We cannot rule out the effect that secreted Wnt pro- CRC progression. Wnt/b-catenin signalling is essential in teins may have on other stromal cell types in healthy and both these processes. Although the source and distribution of tumoural tissue, which warrants further investigation. Wnt proteins in the intestine is still a matter of debate, stro- We compared the list of genes regulated by Wnt3A in mal fibroblasts are thought to be a major source of Wnts.33,34 human colon myofibroblasts with the gene sets representing Here we show that human colon myofibroblasts are highly the biological functions enriched in the expression patterns responsive to Wnt3A, which causes a profound change in associated to each of the four consensus molecular subtypes their gene expression profile leading to the up and downregu- in colorectal cancer (CMS 1–4).8 The pattern of expression of lation of a large set of genes. Therefore, Wnt factors can act Wnt3A-treated myofibroblasts clusterises closest to the on epithelial cells at the bottom of the colon crypt but also molecular subtype CMS2 (Supporting Information Fig. S7A), on pericryptal fibroblasts that harbour functional Wnt recep- which is characterized by WNT and MYC pathway activa- tors and possibly other stromal cell types. The fact that tion.8 Therefore, although myofibroblasts are stromal cells, inhibitors of the Wnt/b-catenin pathway such as DKK2, their molecular signature in response to Wnt3A is closest to NKD1 and 2,orAPCDD1 are among the strongest Wnt3A- that of CMS2 instead of CMS4, despite the latter is character- induced genes in colon myofibroblasts could unveil a mecha- ized by a high stromal component. We also found a signifi- nism to limit the actions of Wnt signalling on these cells or cant enrichment of TCF7L2/TCF-4 binding sites identified in perhaps to protect them from the effects of these factors that LS174T colon cancer cells35 in genes up and downregulated

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Niell et al. 801

Figure 5. An enhancer sequence located 20 kb upstream from the PKP2 transcription start site harbours a functional b-catenin/TCF respon- Molecular Cancer Biology sive element. (a) Diagram of the PKP2 enhancer constructs used in this study showing the location of the putative wild-type (50-CTTTG[A/ T][A/T]-30, white boxes) or mutant (50-CTTTGGC-30, X-crossed white boxes) b-catenin/TCF binding sites. (b) HEK-293T cells were co- transfected with either the forward (–20666/–20091) or reverse (–20091/–20666) 575 bp PKP2 enhancer construct together with TCF4- VP16 (1) or an empty vector (–). The DKK1 promoter was used as a positive control and pRL-TK as an internal control. Cells were lysed 48 hr after transfection and Firefly and Renilla luciferase activities were measured. Values were normalized to those of Renilla. Error bars repre- sent S.D. (***p 0.001). (c) HEK-293T cells were co-transfected with the indicated PKP2 enhancer wild-type or mutant constructs together with TCF4-VP16 (1) or an empty vector (–) and the experiment was performed as in (b). Error bars represent S.D. (n.s., nonsignificant; *** p 0.001). (d) A diagram summarizing the human PKP2 gene regulatory sequences found in this study. White boxes represent functional b-catenin/TCF binding sites. by Wnt3A in CCD-18Co myofibroblasts (Supporting Infor- some conservation among genes regulated by Wnt3A in mation Fig. S7B), which suggests that in spite of their differ- fibroblasts. ent linages there is a set of genes regulated by Wnt proteins One of the genes that were most strongly upregulated by in both cell types. GSEA analysis also showed an enrichment Wnt3A was PKP2, which encodes the desmosomal protein (almost significant) of genes upregulated by Wnt3A in Plakophilin-2. Although this protein is best-known as a com- NIH3T336 (FDR q-val 5 0.08; FWER p-val 5 0.10; Supporting ponent of epithelial cell , it is also involved in Information Fig. S7C) and dermal fibroblasts37 (FDR q- other types of adhesion structures, including composite junc- val 5 0.08; FWER p-val 5 0.18; Supporting Information Fig. tions (area compositae) in cardiomyocytes and a very specific S7D) among genes induced by Wnt3A in CCD-18Co cells. class of Plakophilin-2-containing adherens junctions (coniunc- Likewise, genes downregulated by Wnt3A in CCD-18Co tiones adhaerentes) present in mesenchymally derived cells myofibroblasts were significantly enriched in genes repressed with high proliferative activity.38 Moreover, Plakophilin-2 by Wnt3A in NIH3T336 (FDR q-val 5 0.003; FWER p- seems to have additional functions that are unrelated to val 5 0.002; Supporting Information Fig. S7E) which suggests adhesion, since it has been consistently found inside the cell

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC 802 PKP2 is a novel Wnt/b-catenin target gene in fibroblasts

Figure 6. Plakophilin-2 antagonizes b-catenin/TCF transcriptional activity. (a) HEK-293T cells were transfected with reporter plasmids (pTOP- FLASH or pFOPFLASH) to evaluate the transcriptional activity of the Wnt/b-catenin pathway, together with an expression vector for b-catenin (1) and in the presence or absence of an expression plasmid for Plakophilin-2. pRL-TK was used as an internal control. Cells were lysed 48 hr after transfection and Firefly and Renilla luciferase activities were measured. Values were normalized to those of Renilla, related to those of the mutant pFOPFLASH construct, and represented as fold over control. Error bars represent S.D. (***p 0.001). (b) HEK-293T cells were transfected with reporter plasmids (4xwtCBF1Luc or 4xmtCBF1Luc) to evaluate the transcriptional activity of the Notch pathway, together with an expression vector for IntraCellular Notch Domain (ICND, 1) and in the presence or absence of an expression plasmid for Plakophilin-2. The experiment was performed as in (a). (c) HEK-293T cells were transfected with a reporter plasmid (NF3) to evaluate the

Molecular Cancer Biology transcriptional activity of the NF-jB pathway, together with an expression vector for p65 (1) and in the presence or absence of an expres- sion plasmid for Plakophilin-2. Samples were processed as in (a), values were normalized to those of Renilla, and represented as fold over control. Error bars represent S.D. (*p 0.05). (d) HEK-293T cells were transfected with the b-catenin/TCF responsive DKK1 promoter, together with an expression vector for b-catenin (1) and in the presence or absence of an expression plasmid for Plakophilin-2. The experi- ment was performed as in (a) and values are represented as fold over control. Error bars represent S.D. (***p 0.001). (e) b-catenin protein levels are not affected by Plakophilin-2 expression. Extracts from the experiment in (d) were analysed by Western blot to rule out the possi- bility that the effects of Plakophilin-2 on b-catenin/TCF-dependent transcription were due to changes in b-catenin levels as a consequence of Plakofilin-2 expression.

nucleus.39,40 We show here that Plakophilin-2 expression in pathway, since SW480 cells harbour a mutant APC protein and HEK-293T cells interferes with b-catenin/TCF transcriptional have constitutively active Wnt signalling. In this regard, multi- activity, which implies that it might also be classified as a potent mesenchymal stromal cells and iPS cells from a patient Wnt inhibitor. Unfortunately, CCD-18Co cells are extremely carrying a PKP2 gene mutation exhibited lower b-catenin/TCF resistant to both transient transfection and lentiviral infec- transcriptional activity than those of a healthy control.42 Inter- tion, so this has precluded Plakophilin-2 gain- and loss-of- estingly, another plakophilin family member, Plakophilin-1, has function studies in these cells. been recently shown to be a critical mediator of Wnt signalling Our results contrast with those of Chen and colleagues who in tooth development and ameloblast differentiation.43 This showed that Plakophilin-2 can bind b-catenin, and that expres- hints at a close link between the PKP family and Wnt/b-catenin sion of Plakophilin-2 in SW480 colon carcinoma cells had a signalling. rather small but positive effect on b-catenin/TCF activity.41 Our data show that PKP2 is regulated by b-catenin This discrepancy suggests that the effect of Plakophilin-2 on b- through several response elements in the promoter and catenin-mediated transcription might be cell-type specific enhancer regions of the gene. Sequence analysis of the puta- or, perhaps, dependent on the status of the Wnt/b-catenin tive b-catenin/TCF binding sites showed that the integrity of

Int. J. Cancer: 142, 792–804 (2018) VC 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Niell et al. 803 the core sequence 50-CCTTTG(A/T)(A/T)-30 was essential for fibroblasts of different origins. This suggests that the gene is functionality. Alterations in the first 50 cytidine (sites #3 and not specifically induced in CAF, but is a general Wnt target #7 in the promoter) still rendered a partially functional bind- in fibroblasts. ing site, but changes affecting one of the 30 adenosines/thy- Plakophilin-2 is the only plakophilin expressed in the midines (sites #1, #2, #5 and #6 in the promoter and sites B heart and is essential for cell-cell adhesion in cardiomyo- and C in the enhancer region) rendered non-functional ele- cytes.50,51 Actually, PKP2 mutations are the leading cause ments. ChIP-seq data obtained from UCSC Genome Browser of arrhythmogenic right ventricular cardiomyopathy confirmed that these sites are functional in vivo, and there- (ARVC):52,53 a major cause of sudden cardiac death. There- fore that the PKP2 gene is a bona fide Wnt target gene. fore, further studies should determine whether PKP2 is also a There is growing evidence of the involvement of Wnt target in the heart and, if so, evaluate the relevance of Plakophilin-2 and other plakophilins and desmosomal pro- its Wnt-regulated expression. 44 teins in cancer. Plakophilin-2 is widely expressed in carci- In summary, we show here that the PKP2 gene encoding 45,46 nomas and in some of them its expression is associated Plakophilin-2 is a novel Wnt/b-catenin target that could 47,48 with invasive and metastatic behaviour. Recently, function as a feedback inhibitor of b-catenin/TCF transcrip- Plakophilin-2 has been shown to promote tumour develop- tional activity in colonic fibroblasts, and perhaps in other cell ment by binding the epidermal growth factor receptor types. This is highly relevant, due to increasing evidence sug- (EGFR) and enhancing its ligand-dependent and independent gesting the involvement of Plakophilin-2 in cancer and other 32 activity. In spite of these data, further research is necessary pathologies, and the key role of Wnt/b-catenin signalling in to clarify the role of Plakophilin-2 in different types of can- colorectal carcinogenesis. cer. Classically, colorectal carcinoma cells have been thought to be refractory to extracellular Wnt factors, since most of Acknowledgements them carry intracellular mutations (APC, CTNNB1/b-catenin We thank Keith Brennan and Manuel Fresno for reagents and Lucille Banham and AXIN1) that result in constitutive activation of the path- for her valuable assistance in the preparation of the English manuscript. This way. However, it has been proposed recently that they can work was supported by the Spanish Ministerio de Economıa y Competitividad respond to exogenous and autocrine Wnt ligands that further and the European Regional Development Fund (Fondos FEDER) (SAF2013– activate the Wnt/b-catenin signalling pathway.49 In addition, 43468-R, and SAF2014–53819-R), the Agencia Estatal de Investigacion-Fondos FEDER (SAF2016–76377-R, AEI/FEDER, UE), the Comunidad de Madrid Wnt factors may exert some of their effects on CRC by tar- (S2010/BMD-2344 Colomics2), the Instituto de Salud Carlos III–Fondos geting CAF. Nevertheless, we found that PKP2 expression is FEDER (RD12/0036/0021, and CIBERONC-CB16/12/00273) and the Nuclear upregulated by Wnt3A in both normal and cancer-associated Receptors Network (Nurcamein, SAF2015–71878-REDT).

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